X-Message-Number: 1390
Date: 03 Dec 92 06:54:37 EST
From: Paul Wakfer <>
Subject: CRYONICS: Freezing Damage (Darwin) Part 2


     Perfusion  of both groups of animals was begun by carrying out  a 
total body washout (TBW) with the base perfusate in the absence of any 
cryoprotective agent.  In the FGP group washout was achieved within  2 
-  3 minutes of the start of open circuit asanguineous perfusion at  a 
flow rate of 160 to 200 cc/min and an average perfusion pressure of 40 
mmHg.   TBW  in  the  FGP  group  was  considered  complete  when  the 
hematocrit  was  unreadable and the venous effluent was  clear.   This 
typically was achieved after perfusion of 500 cc of perfusate. 

     Complete blood washout in the FIGP group was virtually impossible 
to  achieve (see "Results" below).  A decision was made prior  to  the 
start  of  this  study (based on  previous  clinical  experience  with 
ischemic human cryonic suspension patients) not to allow the  arterial 
pressure  to  exceed  60  mmHg for any  significant  period  of  time.  
Consequently, peak flow rates obtained during both total body  washout 
and subsequent glycerol perfusion in the FIGP group were in the  range 
of 50-60 cc/min at a mean arterial pressure of 50 mmHg.

     Due to the presence of massive intravascular clotting in the FIGP 
animals  it  was necessary to delay placement of the  atrial  (venous) 
cannula (lest the drainage holes become plugged with clots) until  the 
large  clots present in the right heart and the superior and  inferior 
vena  cava  had been expressed through the atriotomy.  The  chest  was 
kept  relatively  clear of fluid/clots by active suction  during  this 
interval.   Removal  of  large clots and reasonable  clearing  of  the 
effluent  was usually achieved in the FIGP group after 15  minutes  of 
open  circuit asanguineous perfusion, following which the circuit  was 
closed and the introduction of glycerol was begun.

     The  arterial pO2 of animals in both the FGP and FIGP groups  was 
kept  between  600  mmHg and 760 mmHg throughout  TBW  and  subsequent 
glycerol  perfusion.  Arterial pH in the FGP animals was  between  7.1 
and  7.7  and was largely a function of the degree of  diligence  with 
which  addition of buffer was pursued.  Arterial pH in the FIGP  group 
was 6.5 to 7.3.  Two of the FIGP animals were not subjected to  active 
buffering during perfusion and as a consequence recovery of pH to more 
normal  values  from the acidosis of ischemia (starting  pH  for  FIGP 
animals was typically 6.5 to 6.6) was not as pronounced.

     Introduction  of glycerol was by constant rate addition  of  base 
perfusate  formulation  made up with 6M glycerol  to  a  recirculating 
reservoir  containing 3 liters of glycerol-free base  perfusate.   The 
target  terminal tissue glycerol concentration was 3M and  the  target 
time  course for introduction was 2 hours.  The volume of 6M  glycerol 
concentrate  required  to  reach  a  terminal  concentration  in   the 
recirculating   system  (and  thus  presumably  in  the  animal)   was 
calculated as follows:

     Mc = --------- Mp
           Vc + Vp


     Mc = Molarity of glycerol in animal and circuit.

     Mp = Molarity of glycerol concentrate.

     Vc = Volume of circuit and exchangeable volume of animal.*

     Vp = Volume of perfusate added.

     * Assumes an exchangeable water volume of 60% of the preperfusion      
weight of the animal.

     Glycerolization  of  the FGP animals was carried out at  10*C  to 
12*C.   Initial  perfusion  of FIGP animals was at  4*C  to  5*C  with 
warming  (facilitated  by  TBW with warmer perfusate  and  removal  of 
surface  ice packs) to 10*-12*C for cryoprotectant introduction.   The 
lower  TBW  temperature of the FIGP animals was a consequence  of  the 
animals  having  been refrigerated on ice for the 24  hours  preceding 

     Following  termination  of the cryoprotective ramp,  the  animals 
were  removed  from bypass, the aortic cannula was left  in  place  to 
facilitate  prompt reperfusion upon rewarming, and the venous  cannula 
was removed and the right atrium closed.  The chest wound was  loosely 
closed using surgical staples.

     Concurrent with closure of the chest wound, a burrhole craniotomy 
3  to  5  mm in diameter was made in the right parietal  bone  of  all 
animals  using a high speed Dremel "hobby" drill.  The purpose of  the 
burrhole  was  to  allow for  post-perfusion  evaluation  of  cerebral 
volume, assess the degree of blood washout in the ischemic animals and 
facilitate  rapid expansion of the burrhole on rewarming to allow  for 
the visual evaluation of post-thaw reperfusion (using dye).  

     The  rectal  thermistor probe used to  monitor  core  temperature 
during  perfusion was replaced by a copper/constantan thermocouple  at 
the  conclusion  of perfusion for monitoring of the  core  temperature 
during cooling to -79*C and -196*C.

Cooling to -79*C

     Cooling  to -79*C was carried out by placing the  animals  within 
two 1 mil polyethylene bags and submerging them in an isopropanol bath 
which  had  been  precooled to -10*C.   Bath  temperature  was  slowly 
reduced  to  -79*C  by the periodic addition of dry  ice.   A  typical 
cooling curve obtained in this fashion is shown in Figure 5.   Cooling 
was at a rate of approximately 4*C per hour.

Cooling to and Storage at -196*C

     Following cooling to -79*C, the plastic bags used to protect  the 
animals  from  alcohol were removed, the animals  were  placed  inside 
nylon  bags with draw-string closures and were then positioned atop  a 
6" high aluminum platform in an MVE TA-60 cryogenic dewar to which 2"-
3" of liquid nitrogen had been added.  Over a period of  approximately 
48  hours  the liquid nitrogen level was gradually  raised  until  the 
animal  was  submerged.  A typical cooling curve  to  liquid  nitrogen 
temperature  for animals in this study is shown in Figure 6.   Cooling 
rates to liquid nitrogen temperature were approximately 2*C per  hour.  
After  cool-down  animals  were maintained in liquid  nitrogen  for  a 
period  of  6-8  months until being removed  and  rewarmed  for  gross 
structural, histological, and ultrastructural evaluation.


     The  animals  in  both groups were rewarmed to -2*C  to  -3*C  by 
removing them from liquid nitrogen and placing them in a precooled box 
insulated on all sides with a 2" thickness of styrofoam and containing 
a small quantity of liquid nitrogen.  The animals were then allowed to 
rewarm to approximately -20*C, at which time they were transferred  to 
a  mechanical  refrigerator at a temperature of 8*C.   When  the  core 
temperature  of the animals had reached -2*C to -3*C the animals  were 
removed to a bed of crushed ice for post-mortem examination and tissue 
collection  for  light and electron microscopy.  A  typical  rewarming 
curve is presented in Figure 7.

Modification of Protocol Due To Tissue Fracturing

     After the completion of the first phase of this study  (perfusion 
and  cooling  to  liquid nitrogen temperature)  the  authors  had  the 
opportunity  to evaluate the gross and histological condition  of  the 
remains  of three human cryonic suspension patients who  were  removed 
from  cryogenic  storage  and  converted  to  neuropreservation  (thus 
allowing  for post-mortem dissection of the body, excluding the  head) 
(10).  The results of this study confirmed previous, preliminary, data 
indicative of gross fracturing of organs and tissues in animals cooled 
to  and  rewarmed from -196*C.  These findings led us to  abandon  our 
plans  to  reperfuse  the  animals  in  this  study  with  oxygenated, 
substrate-containing  perfusate  (to have been  followed  by  fixative 
perfusion  for histological and ultrastructural evaluation) which  was 
to be have been undertaken in an attempt to assess post-thaw viability 
by  evaluation  of post-thaw oxygen consumption, glucose  uptake,  and 
tissue-specific enzyme release.

     Rewarming  and  examination  of the first  animal  in  the  study 
confirmed  the presence of gross fractures in all organ systems.   The 
scope  and severity of these fractures resulted in disruption  of  the 
circulatory system, thus precluding any attempt at reperfusion as  was 
originally planned.

Preparation of Tissue Samples For Microscopy


     Samples of four organs were collected for subsequent histological 
and  ultrastructural  examination:  brain, heart,  liver  and  kidney.  
Dissection  to  obtain  the tissue samples was begun as  soon  as  the 
animals  were  transferred to crushed ice.  The brain  was  the  first 
organ  removed  for sampling.  The burrhole created at  the  start  of 
perfusion  was  rapidly extended to a full craniotomy  using  rongeurs 
(Figure  8).   The  brain was then removed en bloc to  a  shallow  pan 
containing  iced,  modified Karnovsky's fixative  containing  25%  w/v 
glycerol  (see  Table  I  for composition)  sufficient  to  cover  it.  
Slicing of the brain into 5 mm thick sections was carried out with the 
brain  submerged  in fixative in this manner.  At  the  conclusion  of 
slicing  a 1 mm section of tissue was excised from the  visual  cortex 
and  fixed  in a separate container for electron  microscopy.   During 
final  sample  preparation for electron microscopy care was  taken  to 
avoid  the  cut  edgdes  of the tissue block  in  preparing  the  Epon 
embedded sections.

     The  sliced  brain  was  then placed in  350  ml  of  Karnovsky's 
containing  25%w/v glycerol in a special stirring apparatus  which  is 
illustrated  in Figure 9.  This  fixation/deglycerolization  apparatus 
consisted of two plastic containers nested inside of each other atop a 
magnetic stirrer.  The inner container was perforated with numerous  3 
mm holes and acted to protect the brain slices from the stir bar which 
continuously  circulated the fixative over the slices.   The  stirring 
reduced  the likelihood of delayed or poor fixation due to overlap  of 
slices  or stable zones of tissue water stratification.   (The  latter 
was a very real possibility owing to the high viscosity of the  25%w/v 
glycerol-containing Karnovsky's.)

Deglycerolization of Samples 
     To avoid osmotic shock all tissue samples were initially immersed 
in Karnovsky's containing 25%w/v glycerol at room temperature and were 
subsequently  deglycerolized  prior  to  staining  and  embedding   by 
stepwise    incubation    in   Karnovsky's    containing    decreasing 
concentrations  of  glycerol  (see  Figure  10  for  deglycerolization 

     To  prepare  tissue sections from heart, liver,  and  kidney  for 
microscopy,  the  organs  were  first removed  en  bloc  to  a  beaker 
containing an amount of ice-cold fixative containing 25% w/v  glycerol 
sufficient  to cover the organ.  The organ was then removed to a  room 
temperature  work  surface at where 0.5 mm sections were made  with  a 
Stadie-Riggs microtome.  The microtome and blade were pre-wetted  with 
fixative,  and cut sections were irrigated from the microtome  chamber 
into  a beaker containing 200 ml of room-temperature fixative using  a 
plastic  squeeze-type  laboratory  rinse  bottle  containing  fixative 
solution.   Sections  were  deglycerolized using  the  same  procedure 
previously detailed for the other slices. 

Osmication and Further Processing

     At  the  conclusion  of deglycerolization of  the  specimens  all 
tissues  were  separated into two groups; tissues to be  evaluated  by 
light microscopy, and those to be examined with transmission  electron 
microscopy.   Tissues for light microscopy were shipped  in  glycerol-
free  modified  Karnovsky's solution to American  Histolabs,  Inc.  in 
Rockville,  MD  for  paraffin  embedding,  sectioning,  mounting,  and 

     Tissues   for  electron  microscopy  were  transported   to   the 
facilities  of the University of California at San Diego in  glycerol-
free  Karnovsky's at 1* to 2*C for osmication, Epon embedding, and  EM 
preparation of micrographs by Dr. Paul Farnsworth.

     Due  to  concerns  about the osmication and  preparation  of  the 
material processed for electron microscopy by Farnsworth, tissues from 
the  same  animals  were also submitted  for  electron  microscopy  to 
Electronucleonics of Silver Spring, Maryland.

***Electronucleonics  results  are  not  covered  here  since  another 
investigator  has yet to provide the necessary information and  we  do 
not have access to the pictures. 


Perfusion of FGP Animals 

     Blood  washout  was  rapid and complete in the  FGP  animals  and 
vascular  resistance  decreased  markedly  following  blood   washout.  
Vascular  resistance increased steadily as the glycerol  concentration 
increased,  probably  as a result of the increasing viscosity  of  the 

     Within   approximately  5  minutes  of  the  beginning   of   the 
cryoprotective ramp, bilateral ocular flaccidity was noted in the  FGP 
animals.   As  the perfusion proceeded, ocular  flaccidity  progressed 
until  the  eyes had lost approximately 30% to 50%  of  their  volume.  
Gross  examination  of the eyes revealed that initial water  loss  was 
primarily  from the aqueous humor, with more significant  losses  from 
the posterior chamber of the eyes apparently not occurring until later 
in  the  course  of  perfusion.  Within 15 minutes  of  the  start  of 
glycerolization  the corneal surface became dimpled and irregular  and 
the eyes had developed a "caved-in" appearance.

     Dehydration  was also apparent in the skin and  skeletal  muscles 
and  was  evidenced  by  a marked decrease  in  limb  girth,  profound 
muscular  rigidity,  cutaneous  wrinkling (Figure 11),  and  a  "waxy-
leathery" appearance and texture to both cut skin and skeletal muscle.  
Tissue water evaluations conducted on ileum, kidney, liver, lung,  and 
skeletal  muscle  confirmed  and  extended  the  gross   observations.   
Preliminary  observation suggest that water loss was in the  range  of 
30%  to 40% in most tissues. As can be seen in Table III,  total  body 
water  losses  attributable  to dehydration, while  typically  not  as 
profound, were still in the range of 18% to 34%.  The gross appearance 
of  the heart suggested a similar degree of dehydration, as  evidenced 
by modest shrinkage and the development of a "pebbly" surface  texture 
and a somewhat translucent or "waxy" appearance.

     Examination  of  the cerebral hemispheres through the  burr  hole 
(Figure  12)  revealed an estimated 30% to 50% reduction  in  cerebral 
volume,  presumably  as a result of osmotic dehydration  secondary  to 
glycerolization.   The cortices also had the "waxy"  amber  appearance 
previously observed as characteristic of glycerolized brains.

     The  gross  appearance  of the kidneys,  spleen,  mesenteric  and 
subcutaneous  fat, pancreas, and reproductive organs  (where  present) 
were   unremarkable.   The  ileum  and  mesentery  appeared   somewhat 
dehydrated,  but  did  not  exhibit  the  waxy  appearance  that   was 
characteristic of muscle, skin, and brain.

     Oxygen  consumption (determined by measuring the  arterial/venois 
difference)  throughout  perfusion  was fairly constant  and  did  not 
appear to be significantly impacted by glycerolization, as can be seen 
Figure 12.

Perfusion of FIGP Animals

     As previously noted, the ischemic animals had far lower flowrates 
at  the  same  perfusion  pressure as  FGP  animals  and  demonstrated 
incomplete  blood  washout.   Intravascular  clotting  was  serious  a 
barrier  to  adequate perfusion.   Post-thaw  dissection  demonstrated 
multiple  infarcted areas in virtually all organ systems; areas  where 
blood  washout  and  glycerolization were incomplete  or  absent.   In 
contrast  to  the even color and texture changes observed in  the  FGP 
animals,  the  skin of the FIGP animals  developed  multiple,  patchy, 
nonperfused   areas  which  were  clearly  outlined  by   surrounding, 
dehydrated, amber-colored glycerolized areas.  

     External  and internal examination of the brain and  spinal  cord 
revealed  surprisingly  good  blood washout  of  the  central  nervous 
system.  While grossly visible infarcted areas were noted, these  were 
relatively  few  and  were generally no larger than 2 mm to  3  mm  in 
diameter.   With few exceptions, the pial vessels were free  of  blood 
and appeared empty of gross emboli.  One striking difference which was 
consistently  observed  in  FIGP  animals  was  a  far  less  profound 
reduction  in brain volume during glycerolization (Figure  13).   This 
may  have  been due to a number of factors: lower flow  rates,  higher 
perfusion  pressures,  and the increased  capillary  permeability  and 
perhaps increased cellular permeability to glycerol.  

     Whereas   edema   was   virtually   never   a   problem    during 
glycerolization  of  FGP  animals, edema was  universal  in  the  FIGP 
animals  after as little as 30 minutes of perfusion.  In  the  central 
nervous  system this edema was evidenced by a "rebound"  from  initial 
cerebral  shrinkage  to  frank  cerebral  edema,  with  the  cortices, 
restrained by the dura, often abutting or slightly projecting into the 
burrhole.   Marked  edema of the nictating membranes,  the  lung,  the 
intestines,  and  the  pancreas  was also a  uniform  finding  at  the 
conclusion  of cryoprotective perfusion.  The development of edema  in 
the central nervous system sometimes closely paralleled the  beginning 
of "rebound" of ocular volume and the development of ocular turgor and 
frank ocular edema.  

     In contrast to the relatively good blood washout observed in  the 
brain,  the  kidneys  of  FIGP animals had a  very  dark  and  mottled 
appearance.   While  some  areas (an estimated  20%  of  the  cortical 
surface) appeared to be blood-free, most of the organ remained  blood-
filled throughout perfusion.  Smears of vascular fluid made from renal 
biopsies  which  were collected at the conclusion  of  perfusion  (for 
tissue  water determinations) revealed the presence of many  free  and 
irregularly clumped groups of crenated and normal-appearing red cells, 
further evidence of the incompleteness of blood washout.   Microscopic 
examination  of recirculating perfusate revealed some free, and a  few 
clumped  red  cells.   However, the concentration  was  low,  and  the 
perfusate  microhematocrit  was  unreadable  at  the  termination   of 
perfusion (i.e., less than 1%).

     The  liver  of  FIGP  animals  appeared  uniformly   blood-filled 
throughout  perfusion,  and  did not exhibit even  the  partial  blood 
washout evidenced by the kidneys.  However, despite the absence of any 
grossly  apparent blood washout, tissue water evaluations in one  FIGP 
animal  were  indicative  of  osmotic dehydration  and  thus  of  some 

     The mesenteric, pancreatic, splanchic, and other small  abdominal 
vessels  were  largely free of blood by the conclusion  of  perfusion.  
However,  blood-filled  vessels  were not  uncommon,  and  examination 
during   perfusion   of   mesenteric   vessels   performed   with   an 
ophthalmoscope  at 20X magnification revealed stasis in  many  smaller 
vessels, and irregularly shaped small clots or agglutinated masses  of 
red  cells in most of the mesenteric vessels.   Nevertheless,  despite 
the   presence  of  massive  intravascular  clotting,  perfusion   was 
possible, and significant amounts of tissue water appear to have  been 
exchanged for glycerol.

     One  immediately  apparent difference between the  FGP  and  FIGP 
animals  was  the  accumulation in the lumen of  the  ileum  of  large 
amounts  of  perfusate  or perfusate  ultrafiltrate  by  the  ischemic 
animals.  Within approximately 10 minutes of the start of reperfusion, 
the  ileum  of the ischemic animals that had  been  laparotomized  was 
noticed  to  be  accumulating fluid.  By the  end  of  perfusion,  the 
stomach  and the small and large bowel had become massively  distended 
with  perfusate.   Figure  14 shows both FIGP and  FGP  ileum  at  the 
conclusion  of glycerol perfusion.  As can be clearly seen,  the  FIGP 
intestine  is markedly distended.  Gross examination of the  gut  wall 
was   indicative  of  tissue-wall  edema  as  well   as   intraluminal 
accumulation  of  fluid.  Often by the end of perfusion, the  gut  had 
become  so  edematous  and  distended  with  perfusate  that  it   was 
impossible  to completely close the laparotomy  incision.   Similarly, 
gross  examination of gastric mucosa revealed severe erosion with  the 
mucosa being very friable and frankly hemorrhagic.

     Escape  of  perfusate/stomach contents from the  mouth  (purging) 
which occurs during perfusion in ischemically injured human suspension 
patients did not occur, perhaps due to greater post-mortem  competence 
of the gastroesophageal valve in the cat.

     Oxygen  consumption  in  the two ischemic cats in  which  it  was 
measured  was dramatically impacted, being only 30% to 50% of  control 
and deteriorating throughout the course of perfusion (Figure 12).

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